Enteroaggregative E. coli (EAggEC) are a recently described category of enteric pathogen associated with acute and, especially, persistent pediatric in the developing world; persistent diarrhea is a disease of increasing public health importance. The broad, long term objectives of this project are genetic, pathogenetic, physical and epidemiologic characterization of the plasmid-associated aggregative adherence (AA) factor in enteroaggregative E. coli (Eaggec). The ultimate objective will be to evaluate the possibility of using the AA factor as an EAggEC vaccine. The central hypothesis to be tested by this proposal is that all EAggEC share a common AA adhesin essential for full virulence. The specific aims of this proposal are 1) genetic characterization of the AA factor, 2) construction of isogenic AA-deficient mutants, 3) assessment of the genetic control of AA, 4) determination of the physical structure conferring AA, 5) evaluation of the role of AA in pathogenesis, and 6) development of improved diagnostic methodology based on AA gene detection. The experimental design involves cloning the genes. encoding the AA factor from strain 17-2, localization of all relevant regions by insertion mutagenesis, and sequencing of AA genes. Gene products will be expressed under the control of T7 promoter sequences. Isogenic insertion and, deletion mutants will be constructed which are deficient in AA; these will be used to identify AA-related surface structures and the role of AA in causing intestinal histopathology in animal models and cell culture. Oligonucleotide probes will be used to determine the prevalence of the AA factor among EAggEC and other E. coli. Methods employed in this proposal include cosmid cloning of AA- regions, chain termination M13 sequencing of cloned regions and T7 transcription/translation of cloned gene products. Isogenic mutants will be constructed by introducing deletions into the AA genes which do not interrupt reading frame, then reintroducing these mutations into the native genome by homologous recombination. Ultrastructure of AA will be examined using cells in culture. Isogenic mutants will be tested in ligated rabbit intestinal loop and gnotobiotic piglet models for loss of virulence. Genetic regulation and cellular localization of gene products will be studied via construction of TnphoA and TnlacZ gene fusions.